The present invention is directed to increasing resveratrol concentrations in peanuts and peanut products.
The antioxidative activity (AOA) provided by phenolic compounds has been shown to inhibit the oxidation of low density proteins (2), thereby decreasing heart disease risks (3). Phenolic compounds have also been shown to have anti-inflammatory (4) and anti-carcinogic activity (5).
Resveratrol (trans-3,5,4′-trihydroxystilbene), a stilbene phytoalexin, is a phenolic compound possessing antioxidant activity. Resveratrol has been shown to provide health-promoting activities such as lowering the incidence of coronary heat disease (6), provides cancer chemopreventive activity (7) and is a phytoestrogen exhibiting variable degrees of estrogen receptor agonism (8).
Resveratrol can be found in two isomeric forms trans- and cis-resveratrol. Trans-resveratrol is the isomer most abundantly found in nature. However trans-resveratrol is transformed into the cis-isomer after exposure to UV light. Production of resveratrol in plants occurs as a defense response to exterior stress.
Various biotic and abiotic treatments have been shown to increase AOA, total phenolic compounds, and resveratrol concentration in plant material. Biotic factors such as cultivar type (9-11), maturity level (9-11) and microbial exposure have been tested for their elicitation effect on peanuts. In addition abiotic factors such as wounding by slicing in peanut (11, 17, 18), ultraviolet (UV) light exposure on grape and peanut leaves (15, 16, 19-21), ultrasound (22) exposure applied to Panax ginseng cells, and processing methods, such as roasting of peanuts (23), have also been studied as elicitors. Among these methods only the effect of slicing on resveratrol synthesis was studied on peanut kernels.
In one study, the AOA of methanolic extracts of peanut hulls was found to be 94.8 to 93.9% in peanuts harvested at 74 to 144 d after planting (9). In a later study (10), the AOA of methanolic extracts of peanut hulls from Spanish, Valencia, and Runner, and Virginia cultivars were found to be 96.1, 96.8, 96.1 and 96.6%, respectively. These studies show that neither maturity level (9) nor cultivar (10) significantly affects the AOA of methanolic extracts of peanut hulls.
In another study, UV light held at a distance of 110 mm from the surface of methanolic extracts of peanut hull powder for 0, 3, and 6 days resulted in an AOA of 99.7, 96.5, and 96.5%, respectively (24). The findings indicated that UV light had no significant effect on AOA (24). In yet another study, Hwang et al. (23) showed that peanuts roasted at 180° C. for up to 60 min provided remarkable AOA for linoleic acid in emulsion and their antioxidative effect relatively increased with roasting time from 10 to 60 min.
The total phenolic compound concentration of peanut kernels has not been reported in the literature. However, the total phenolic compound concentration has been determined in peanut hulls (9, 10) and defatted peanut flour (25). Yen and Duh (10) concluded that the difference in the amount of total phenolic compounds due to cultivar type was negligible, and that the difference was due to maturity, with more mature peanuts having higher concentrations of total phenolics. Duh and Yen (24) determined the effect of UV light exposure on extracts of peanut hull powder. They found that UV light exposure significantly decreased (p<0.05) the amount of total phenolic compounds to 7.80, 7.53 and 7.05 mg/g after exposure to UV light for 0, 3, and 6 d, respectively (24).
Red wine is one of the most common food sources of resveratrol, at an amount of 0.99-5.01 mg/L (26). Trans-resveratrol has also been identified in peanut kernels and processed peanut products. Roasted peanuts contain the lowest content of resveratrol, 0.055±0.023 μg/g, peanut butter contains a significantly higher amount, 0.324±0.129 μg/g, and boiled peanuts have the highest concentration, 5.138±2.849 μg/g (27).
Peanut kernels inoculated with microorganisms are usually sliced (12, 13) or ground (14) prior to treatment. Ingham (12) found that after slicing the ends of peanut kernels, inoculating with Helminthosporium carbonum increased resveratrol from 0 to 38-55 μg/ml after incubation for 24 h at 22° C. Aguamah et al. (13) reported that three phytoalexins increased in peanut kernels soaked in water overnight, sliced, exposed to their natural microflora and incubated. Sobolev et al. (14) later found that fully-imbibed peanut kernels ground into 3-5 mm pieces and inculcated with Aspergillus flavus and A. parasiticus, contained 30 μg/g of resveratrol after incubation.
Microbial exposure has been shown to be an effective elicitor for resveratrol in peanut kernels (12-14). However, such treatment can leave the final product unsafe and inedible. To eliminate microbial influence on the synthesis of resveratrol in peanuts stressed by size-reduction, kernels were surface sterilized with 20% hydrogen peroxide (H2O2) (18) and 5% sodium hypochlorite (11) prior to stress application. Cooksey et al. (17) found surface sterilized (20% H2O2) fully-imbibed peanuts sliced 2 mm thick increased resveratrol concentration. These findings indicate that the occurrence of phytoalexins in peanuts is of potential significance as a defense response against mycotoxigenic fungi of the Aspergillus flavus group (17). Arora and Strange (11) also found that sterilized (5% sodium hypochlorite) fully-imbibed, sliced (1-2 mm) peanut kernels increased resveratrol concentration after incubation for 48 h at 20° C.
Exposure to UV light increased synthesis of resveratrol in grape leaves from 0 to 50-100 μg/g after incubation (16). Grapes exposed to UV light contained 50-233.38 and 150.03-400.08 μg/g, respectively (19). Cantos et al. (20) found that mature grapes exposed for 30 min to UV light at a wavelength of 254 nm and incubated for 10 d at 0° C., followed by 5 additional days at 5° C. synthesize higher resveratrol concentrations of 100 μg/g compared to peanuts exposed to UV light at 340 nm resulting in 65 μg/g of resveratrol.
In a more recent study, Cantos et al. (21) developed and characterized an induction modeling method for resveratrol synthesis using UV irradiation pulses on Napoleon table grapes with industrial applicability. The authors found that grapes exposed to UV light at a distance of 40 cm produced higher resveratrol concentrations than at 20 cm, indicating that the UV signal was too strong at the lower distance and the resveratrol “biosynthetic system” was damaged (21). At a distance of 60 cm the induction of resveratrol was delayed compared to that at 40 cm, making the method less feasible for industrial application. Results also showed that the time to achieve the maximum resveratrol level exponentially decreased versus irradiation power. Final selection of optimum conditions was based on economic criteria and included a wavelength of 254 nm (510 W) for 30 s at a distance of 40 cm, which increased resveratrol from 10 μg/g to 115 μg/g (21).
Ultrasound has not been applied specifically as a resveratrol elicitor; however ultrasound has been successfully used in Panax ginseng cells (power density below 82 mW/cm3, 1-4 min) to stimulate the biosynthesis of a secondary metabolite, ginsenoside saponins (22). Cells exposed to ultrasound at a power density of 3.4, 13.7, 34.1, 61.4 and 81.8 mW/cm3 increased saponin concentration from 0.0436 g/L in control cells to a maximum of 0.747, 0.783, 0.775, 0.830 and 0.640 g/L, respectively, after 14 d at 25° C. (22). This study showed that the total ultrasound emitted (i.e., the product of ultrasound power and exposure time) showed a significant correlation with secondary metabolite production.